Transmissible spongiform encephalopathies (TSEs), or prion diseases, represent a novel molecular mechanism of infectivity, based on the misfolding of a self-protein designated PrPC into a pathological, infectious conformation termed PrPSc. Through this model, PrPSc serves as a template to convert the normal cellular protein (PrPC) into the infectious conformation (PrPSc) in an autocatalytic, self-propagating manner (Aguzzi et al., Annu. Rev. Pathol. (2008) 3:11-40).
Prion diseases affect a number of domestic species causing scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle and chronic wasting disease (CWD) in cervids such as deer and elk (Silveira et al., Curr. Top. Microbiol. Immunol. (2004) 284:1-50). Human manifestations of the prion diseases include Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Sheinker syndrome, and fatal familial insomnia (Collinge, Ann. Rev. Neurosci. (2001) 24:519-550). In the absence of any effective therapies, prion diseases currently have a fatal outcome in all species (Geshwind, Lancet Neurol. (2009) 8:304-306). The infectious component of prion disease (PrPSc) is characterized by increased β-sheet content and extreme structural stability. The unique persistence of this infectious conformation in the environment has severe implications on both disease dynamics and strategies for disease control (Wiggins, Neurochem. Res. (2009) 34:158-68).
Natural transmission of PrPSc within populations often occurs following ingestion of contaminated environmental material (Williams, Veterinary Pathology (2005) 42:530-549). The recycling of contaminated animal materials in feed provides an additional mechanism of transmission specific to livestock (Bradley, Livestock Production Science (1994) 38:5-16). Accordingly, efforts to control prion transmission in livestock have focused on removal of these high-risk animal-based components of feed, thereby reducing animal exposure to infectious PrPSc (Smith et al., Br. Med. Bull. (2003) 66:185-198). While this approach enabled prion diseases of livestock to be sufficiently controlled, environmental contamination and spontaneous disease still enable BSE to persist, albeit at low levels. This highlights the inability of current management tools and practices to completely eliminate the threat of BSE in the food supply.
Although BSE infection of cattle has been the most publicized TSE, chronic wasting disease of cervids has recently emerged as the prion disease of most concern in livestock. CWD first surfaced in Colorado in the 1960s and was later classified as a prion disease in 1978 (Williams, J. Wildl. Dis. (1980) 16:89-98). At the time of initial identification of CWD in wild cervids, the disease was believed to be confined to a small demographic. Since then, CWD endemic areas have undergone dramatic geographical expansion, to include extensive regions of both the United States and Canada. The presence of CWD in both farmed and wild cervids, coupled with the large geographic spread, and uncontrolled transmission of this disease, suggests CWD may be one of the most contagious TSEs (Williams, Veterinary Pathology (2005) 42:530-549). The management of CWD in the wild is also complicated by the free-ranging dynamic of infected populations and the opportunity to overcome species barriers through intermediate species such as crows (verCauteren et al., PLoS one (2012) 7:e45774), rodents (Heisey et al., J. Virol. (2010) 84:210-215), and voles (Nonno et al., PLoS pathogens (2006) 2:e12).
Due to the importance of cervids in the hunting, tourism, and agricultural industries, CWD has the potential for severe economic and human health implications (Saunders et al., Emerg. Infect. Dis. (March 2012) 18(3). There are no confirmed cases of human infection with CWD. This could, however, reflect low transmissibility across species to humans, extensive latency periods, and relatively low levels of human consumption of CWD-infected animals. Importantly, transmission of CWD to humans could occur either through direct transmission to humans through consumption of infected cervid meat or indirectly through infection of a secondary species such as cattle. The potential for disease transmission between cervids and cattle is a possibility, due to the close ecological and phylogenetic relationship of these species (Sigurdson and Agguzi, Biochimica et Biophysica Acta (2007) 772:610-618). Cerebral inoculation of cattle with CWD material results in the development of a TSE (Hamir et al., J. Vet. Diagn. Invest. (2001) 13:91-96). Although this method of infection is quite extreme, the potential for CWD to overcome this species barrier is still a possibility.
Thus, there is a clear need for strategies to prevent and treat prion diseases of humans and animals. There have been numerous studies examining the use of immunotherapy for prion diseases. In this regard, several studies have demonstrated the ability of antibodies against PrPC, or specific fragments of the protein, to offer protection in both in vitro and in vivo models (Enari et al., Proc. Natl. Acad. Sci. USA (2001) 98:9295-9299; Perrier et al., J. Neurochem. (2004) 89:454-463). This includes passive and active immunization as well as engineered expression of PrPC binding fragments (White et al., Nature (2003) 422:80-83; Sigurdsson et al., Neurosci. Lett. (2003) 336:185-187; Sigurdsson et al., Am. J. Pathol. (2002) 161:13-17; Peretz et al., Nature (2001) 412:739-743). However, these studies fell short of providing either a prophylactic or therapeutic prion vaccine.
While these findings are encouraging, a number of practical considerations must be addressed prior to the development of a real-world prion vaccine. One of the main challenges for prion vaccine development is overcoming tolerance to PrPC. There have been a number of investigations that attempted, with varying degrees of success, to overcome self-tolerance and induce strong antibody responses to PrPC protein. These investigations have employed a variety of different carrier systems and adjuvants to induce antibody responses (Hanan et al., Biochem. Biophys. Res. Commun. (2001) 280: 115-120; Koller et al., J. Neuroimmunol. (2002) 132:113-116; Sigurdsson et al., Am. J. Pathol. (2002) 161:13-17; Rosset et al., J. Immunol. (2004) 172: 5168-5174; Polymenidou et al., Proc. Natl. Acad. Sci. USA (2004) 101 Suppl 2:14670-14676; Schwarz et al., Neurosci. Lett. (2003) 350:187-189; Gilch et al., J. Biol. Chem. (2003) 278:18524-18531). Notably, however, many of these investigations resort to harsh adjuvants and vaccination regimens that are impractical for either humans or livestock.
Importantly, the strategy for overcoming self-tolerance must also incorporate a method for limiting antibody reactivity with non-pathogenic conformations of PrPC. Due to the ubiquitous expression of this cell surface protein, the generation of circulating PrPC antibodies may result in a variety of adverse consequences in vivo, both functional and immunological. For example, antibody binding may initiate improper activation of PrPC-based cell signaling cascades (Cashman et al., Cell (1990) 61:185-192; Schneider et al., Proc. Natl. Acad. Sci. USA (2003) 100:13326-13331; Arsenault et al., Prion (2012) 6:477-488), or trigger apoptosis in neurons (Solforosi et al., Science (2004) 303:1514-1516). The activation of a non-discriminating antibody response to PrPC may result in the subsequent activation of complement-dependent cell lysis, facilitated by antibody binding to PrPC at the cell surface, or may facilitate the development of autoimmune disease, by breaking PrPC tolerance. Although the exact physiological role of PrPC has yet to be fully elucidated, ideally, an effective prion vaccine would be specific to PrPSc. The strategy for conformation specific targeting of PrPSc requires the identification of epitope regions that are surface exposed in the infectious misfolded conformation, yet remain concealed in the non-pathogenic isoform.
It has been reported that a YYR motif was specifically exposed upon experimental misfolding of PrPC (Paramithiotis et al., Nature Medicine (2003) 9:893-899). U.S. Pat. No. 7,041,807 describes rabbit polyclonal antisera raised against the YYR peptide and immunoprecipitation of PrPSc from scrapie-infected mouse brain but did not PrPC from uninfected brains. However, the opportunity to translate this epitope into a vaccine was restricted by the minimal immunogenicity of this motif; PrPSc-specific monoclonal antibodies (mAbs) were restricted to IgM isotype after multiple immunizations with Freunds complete adjuvant (Paramithiotis et al., Nature Medicine (2003) 9:893-899). Strategies of formulation and delivery, including presenting the peptide in the context of a potent carrier system designed to facilitate antibody responses to self peptides, still failed to generate epitope specific immune responses. Sequence optimization of the core epitope was essential to generate more immunogenic peptides.
U.S. Patent Publ. 2009/0280125 describes chimeric vaccines representing various expansions around the YYR core. Screening of these vaccines in animals identified expansions that satisfied the criteria of increased immunogenicity while retaining PrPSc specificity. As such, this approach was successful but time consuming and labor intensive.
U.S. Patent Publ. 2012/0107321 describes a second prion disease-specific epitope designated YML. The YML epitope also shows prion-specific exposure, and is not present at the surface of normal cells when probed with antibody and analyzed by flow cytometry.
Despite the above advances, there remains a need for the development of effective strategies for the treatment, prevention and diagnosis of prion infection.